In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

摘要

Yersinia pestis, the agent of plague, encodes several essential virulence factors on a 70 kB plasmid, including the Yersinia outer proteins (Yops) and multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type three secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (Mphis) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 5 h. The interactions were discerned from results of parallel microscopy, immunoblot, and fluorescence-activated cell sorting analyses. The Mphis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies Abs) in conjunction with organelle-specific Abs or dyes. The samples were observed for co-localization by immuno- fluorescence and -electron microscopy. For fractionation studies, uninfected and infected Mphis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Lysates were further purified on immunoaffinity columns treated with biotinylated anti-V. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs.